chaffeensis RNAP and its use in characterizing the transcriptiona

chaffeensis RNAP and its use in characterizing the transcriptional profiles of two p28-Omp gene

(p28-Omp) promoters. In this study, we also described the recombinantly expressed E. chaffeensis sigma factor, σ70, and its use in promoter analysis studies after its reconstitution with E. coli core enzyme. Modulatory effect of E. chaffeensis protein lysates on in vitro transcription is also described in this study to serve as the first step towards determining the regulatory mechanisms underlying gene expression in this pathogen. Results Isolation of E. chaffeensis RNA polymerase (E. chaffeensis RNAP) E. chaffeensis DNA-dependent RNA polymerase (E. chaffeensis RNAP) was partially purified from the organisms grown in macrophage cultures by adapting heparin-agarose column purification method described earlier for other bacterial systems [27]. To determine the purity and polypeptide composition of the E. chaffeensis CH5424802 concentration RNAP, several eluted fractions were electrophoresed ATM inhibitor on a polyacrylamide gel

that was stained using silver nitrate (Figure 1A). The gel pattern revealed that the E. chaffeensis RNAP had a subunit structure similar to E. coli RNAP (that is also typical of other eubacteria) with five major subunits (α2, β, β’, σ). Western blot analysis confirmed the presence of E. chaffeensis σ70 polypeptide when assessed using a heterologous E. coli anti-σ70 monoclonal antibody, 2G10 (Figure 1B). Amino acid alignment of the sequence of E. chaffeensis σ70 polypeptide with E. coli σ70 polypeptide revealed significant homology which also spanned to the putative binding site sequence of 2G10 antibody to E. coli σ70 polypeptide [28, 29] (Figure 2). The homology between amino acid residues of σ70 polypeptides recognised by 2G10 antibody [28] is considerably 4��8C higher between E. chaffeensis and E. coli than between E. chaffeensis and Chlamydia trachomatis . Protein BLAST search (at National Center for Biotechnology Information

Bethesda, MD, USA) of the putative amino acid binding site sequence of 2G10 in E. coli [28, 29] against E. chaffeensis (Arkansas isolate) genome identified only one significant match (E-value of 1e-11 and having 69% identity) with E. chaffeensis RNAP σ70 polypeptide, RpoD. Figure 1 E. chaffeensis RNA polymerase purification by employing heparin agarose column purification method. A) Silver-stained SDS-PAGE gel profile of heparin agarose purified fractions of E. chaffeensis RNA polymerase. M, protein standards (kDa); C, E. chaffeensis crude lysate; W1, first wash fraction from the column; W2, second column wash; E1, first elution fraction; E2, second elution fraction; P, pooled dialyzed fractions of eluted fractions 3 to 6; Ec, E. coli holoenzyme from Epicenter® B) Western blot analysis of the proteins resolved in panel A with E. coli anti-sigma70 monoclonal antibody, 2G10. Figure 2 Comparative alignment of complete amino acid sequences of E. chaffeensis (ECH), E. coli (ECOLI) and C.

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